Cobweb is a fungal disease of commercially cultivated mushrooms. Several members of the ascomycete genus
Cladobotryum
sp.
have been reported as causal agents. White button mushroom is the most frequently cited host, but a wide range of cultivated edible
mushrooms suffer cobweb. The pathology causes production losses and reduces the crop surface available. The parasite produces a
great number of harmful conidia that can be released easily and distributed throughout the mushroom farm to generate secondary points
of infection. To prevent initial outbreaks, hygiene is of primary importance within the facilities dedicated to mushroom cultivation,
while additional measures must be implemented to control and reduce cobweb if there is an outbreak, including chemical and biological
methods. This review summarizes and discusses the knowledge available on the historic occurrence of cobweb and its impact on
commercial mushroom crops worldwide. Causal agents, disease ecology, including the primary source of infection and the dispersal of
harmful conidia are also reviewed. Finally, control treatments to prevent the disease from breaking out are discussed.

Additional key words:Cladobotryum
;
fungal disease;dispersal;production losses;edible mushroom;controlMINECO, Spain (Project RTA2010-00011-C02) and FEDER (Project E-RTA2014-00004-C02). JC was the recipient of
a fellowship from the FPI-INIA program of Ministerio de Economia y Competitividad.

Author´s contributions:
Conception or design; acquisition, analysis, and interpretation of data; drafting of the manuscript; and
coordinating the research project: JC. Critical revision of the manuscript for important intellectual content; administrative, technical, or
material support; and supervising the work: JC, MJN and FJG. Obtaining funding: FJG.

Competing interests:
The authors have declared that no competing interests exist.

Many fungal diseases can affect commercial mushroom crops (
Fletcher & Gaze, 2008
). Among them cobweb is considered one of the most serious diseases for white button mushroom [
Agaricus bisporus
(Lange) Imbach] cultures, the most widely cultivated species (
Royse, 2014
). Other edible cultivated mushrooms may also develop the harmful pathology (
Gea
et al.
, 2011
,
2017
;
Back
et al.
, 2012
;
Kim
et al.
, 2012
). Its occurrence in commercial crops results in reductions in yield and quality, mainly due to cap spotting, a lesser surface area that can be used for cultivation and to the need for early crop termination when the disease becomes epidemic (
Adie, 2000
;
Adie
et al.
, 2006
).

Cobweb appears more often at the end of the crop cycle (although the earlier it appears, the more devastating it can be) during the autumn and winter cycles. First, small, white circular patches appear on the casing soil or basidiomes. These quickly spread by means of a fine grey-white mycelium that resembles a spider web (
Carrasco
et al.,
2016a
). Eventually patches of mycelium start to sporulate, producing masses of dry spores that are easy to release when they are physically disturbed, mainly through watering or picking operations - even air currents from air-conditioning systems are sufficiently strong to mobilize the harmful spores (
Adie
et al.
, 2006
). Once released, conidia are spread throughout the mushrooms facilities by air currents to form secondary colonies on the casing layer or to simultaneously spot the basidiomes (
Adie, 2000
). As soon as a primary cobweb outbreak is located over the casing or carpophores, it must be treated before sporulation, covering the infected area with thick damp paper to avoid the release of conidia and disease dispersion (
Pyck & Grogan, 2015
).

Control methods must be implemented through hygiene measures and by preventing the dissemination of spores, which are dry and easy to dislodge. When not properly treated, conidia will spread within crops, magnifying infection and increasing losses (
Adie
et al.
, 2006
;
Pyck & Grogan, 2015
). In this respect, public policies aimed at reducing the use of chemical pesticides through the use of sustainable agriculture practices (
e.g
. the French “Ecophyto 2018” plan) have led to the intensification of biological control efforts in agriculture. Although there have been attempts to identify biological control agents and environmentally-friendly biomolecules that are effective against fungal diseases in mushroom (
Potocnik
et al.
, 2010
;
Kosanovic
et al.
, 2013
;
Gea
et al.
, 2014
;
Geösel
et al.
, 2014
), no efficient bio-treatment to control cobweb disease has been described. In view of this, control of the pathology still relies on the use of chemical fungicides. However, since the sensitivity of mycoparasites to approved pesticides is gradually diminishing and signals of resistance have been detected (
McKay
et al.
, 1998
;
Gea
et al
., 2005
;
Grogan, 2006
), their use demands judicious management. In short, to optimize integrated disease control, the use of chemicals must be combined with good farming practices and with measures directed towards enhancing hygiene within growing facilities.

Cobweb disease: a recurrent visitor

Cultivated edible mushrooms are susceptible to diseases caused by bacteria, fungi and viruses. Among biotic agents, mycoparasites are responsible for the greatest mushroom crop losses, which have a significant economic impact on industry (
Fletcher & Gaze, 2008
).

Historically treated as a minor disease, cobweb is currently considered one of the four most serious diseases of mushroom crops caused by parasitic fungi, together with dry bubble (
Lecanicillium fungicola
), green mould (
Trichoderma aggressivum
) and wet bubble (
Mycogone perniciosa
) (
Fletcher & Gaze, 2008
). In the mid-1990s, cobweb was reported to be the most serious disease affecting mushroom cultivation in UK and Ireland, where it reached epidemic proportions that involved production losses of up to 40% (
Adie
et al.
, 2006
).

The prevalence of cobweb disease in commercial mushroom crops has been reported to vary between 6.8 and 28% in Indian
A. bisporus
facilities (
Seth & Dar, 1989
;
Bhatt & Singh, 2002
), 33% in Turkey (
Bora & Özaktan, 2000
) and, up to 32% in Spanish commercial button mushroom crops (
Carrasco
et al.
, 2016a
). Its occurrence is associated with final flushes and is conditioned by the season (
McKay
et al.,
1999
;
Adie, 2000
;
Desrumeaux, 2005
). The occurrence and severity of cobweb gradually increases from the first to the third flush. Although it may be established at any time during the year, it is of particular concern in autumn and winter (
Carrasco
et al.
, 2016a
).

Causal agent

Several species belonging to the genus
Cladobotryum
Nees
emend.
(syn.
Dactylium
Nees) can cause cobweb disease in edible mushroom crops (
Table 1
). They correspond to the conidial or asexual stage of species from the genus
Hypomyces
(Fries) L.R.Tulasne (Ascomycota, Hypocreales, Hypocreaceae).

When plated on potato dextrose agar (PDA) these fungi develop a greyish-white mycelium with the reverse side of the plate turning yellow in few days. Usually 2-4 weeks later, the plates acquire a deep red colour (
Fig. 1m,n,o
). This pigment, most probably aurofusarin, is mainly secreted by the hyphae immersed in the growth media (
Põldmaa, 2011
). However, not every
Cladobotryum
species provoking cobweb generates the pigment (
Potocnik
et al.
, 2008
).

Cladobotryum
spp. present verticillated hyphae at the end of which three or four conidiogenous cells, called phialides, are located. Most of the species show a conidial holoblastic ontogeny (in which the apex of the conidiogenous cells is incorporated as part of the generated conidium) through basipetal succession (
Grogan & Gaze, 2000
;
Tamm & Põldmaa, 2013
). Conidia, unicellular in origin, usually show from 1 to 3 septa (2 to 4 cells) (
Desrumeaux, 2005
;
Adie
et al.
, 2006
). They are hyaline, globose to subglobose, bacilliform, cylindrical and often lightly tapered, slightly curved in some cases, with a conspicuous basal hilum in the base. The apex shape and dimensions of the subulate phialides (narrowing towards the apex) vary among species (
Fig. 1a-f
).

Cladobotryum dendroides
(Bull.: Fr.) W. Gams & Hoozem. (syn.
Dactylium dendroides
) has been the species historically associated with cobweb disease (teleomorph:
Hypomyces rosellus
(Alb. & Schwein.:Fr.) Tul.).
In vitro
, it secretes the above described pigment when the strain ages (
Põldmaa, 2011
). It is the only species in the genus characterised by a thin-walled sympodial conidiogenous rachis at the phialide tip that apparently is formed after successive conidia are released (
Tamm & Põldmaa, 2013
). The conidia mostly present 2-3 septa.

Certain species of
Cladobotryum
parasite members of the basidiomycetes group, mostly belonging to the orders Aphyllophorales and Agaricales (
Gams & Hoozeman, 1970
). In addition, some species are found on different substrates, such as bark, decaying wood or leaf litter.

According to literature, casing contamination is frequently considered a source of primary infection (
Fletcher & Gaze, 2008
). Casing materials artificially inoculated with the pathogen reproduced cobweb disease in
A. bisporus
and
P. eryngii
(
Carrasco
et al.
, 2016a
;
Gea
et al.
, 2017
). The presence of the host in the casing layer seems necessary to desensitize the dormant spores and to stimulate their germination and the development of
Cladobotryum
mycelium. In pilot trials, we noted that raw casing material remains healthy after inoculation with a suspension of
C. mycophilum
conidia, while the same casing colonized by
A. bisporus
expressed the disease in every replicate (our unpublished data). Likewise,
L. fungicola
is inhibited by the microflora of the casing layer due to a phenomenon called fungistasis; however, the presence of the host removes the fungistasis to facilitate disease development (
Berendsen
et al.
, 2010
).

Tamm & Poldmaa (2013)
concluded that the endemic species determines the causal agent of cobweb in commercial crops. This seems to indicate that a primary source of infection may be wild specimens infected near the farm. Under unfavorable conditions, particularly when the relative humidity (RH) is low, most
C. dendroides
spores do not survive for long periods. However, the fungus produces microsclerotia, resistant structures that can germinate even when they are stored at 0% RH (
Lane
et al.
, 1991
). High humidity conditions outside the cropping rooms will facilitate survival of the pathogenic conidia and their dispersal through the production area (
Carrasco, 2016
;
Carrasco
et al
., 2016a
).

Conversely, compost is not usually considered to be a primary source of infection. During phase II, compost undergoes a high-temperature pasteurisation process to eliminate pathogens (
Fletcher & Gaze, 2008
). Likewise, spawn or host mycelium is not a source of infection due to high-standard hygiene conditions while the spawn grain is prepared (
Desrumeaux, 2005
;
Fletcher & Gaze, 2008
).

Finally, alternative sources of primary infection are contaminated packages and containers, external visits, vehicles, etc. Contaminated water could be also a source of infection since some mycopathogens, such as
Lecanicillium
and
Mycogone
spores, are known to survive in water for many months.

Brown spots are generated when a single spore lands on the mushroom surface and germinates. These spots usually provoke depression of the cap tissue. From the localized spots a parasitic mycelium emerges that eventually engulfs the whole basidiome. The grey-yellowish spotting is due to the interaction between parasitic mycelium and the host basidiomes; spots progressively discolour the mushroom tissue, which succumbs to wet rot (
Adie, 2000
) (
Fig. 2c
).

Prevention is crucial to precluding the emergence of cobweb and to limiting its impact once installed. Control methods should prevent dispersion of conidia (as previously described), which is the main way of infection (
Adie, 2000
;
Adie & Grogan, 2000
;
Fletcher & Gaze, 2008
).

The end of the crop cycle is a crucial time for removal of any residual disease. Wet conidia of
Cladobotryum
spp. are killed by treatment at 45 ºC for 30 min, but they resist higher temperatures when dry, even up to 100 ºC (
Desrumeaux, 2005
). Similarly, the pathogenic mycelium, which is susceptible to a 15 min, 40 ºC treatment when wet, requires temperatures of 70 ºC for 15 min when dry (
Fletcher & Gaze, 2008
). Consequently,
in situ
thermal disinfection at the end of the crop cycle through “cooking-out” (65-70 ºC for 9-12 hours) is the best way to ensure correct disinfection (
Fletcher & Gaze, 2008
). When not possible, it is advisable to clean the empty facility with water and suitable disinfectants.

It is also possible to prevent disease outbreaks by controlling the humidity and the temperature within mushroom facilities, since few spores of
C. dendroides
can germinate with a RH lower than 85% , while at low temperatures (17 ºC) development and spread of the disease are unlikely (
Desrumeaux, 2005
).

Chemical control

Control of cobweb disease is still highly dependent on routine application of fungicides from several chemical groups: prochloraz-Mn (DMI-fungicide, FRAC code: 3) is the fungicide currently recommended by the European Union to treat cobweb (
Carrasco, 2016
;
FRAC, 2016
). Chlorothalonil (chloronitrile, FRAC code: M5) is also approved for use in France, Poland and Spain. Two DMI fungicides, imazalil (to control green mould disease) and prochloraz-Mn (for cobweb), are licensed for use in Australian mushroom crops. In South Africa, prochloraz-Mn and thiabendazole are the fungicides approved for use in mushroom (
Chakwiya
et al.
, 2015
). Thiabendazole (MBC-fungicide, FRAC code: 1) can be used in USA mushroom crops, as well as chlorothalonil formulates. Recently, metrafenone (benzophenone, FRAC code: U8) has been authorized for use in France to fight cobweb disease (
FRAC, 2016
). Recently, too, metrafenone obtained a temporary approval for use on mushroom crops in Spain. When compared with prochloraz-Mn and chlorothalonil, metrafenone showed higher selectivity towards
C. mycophilum in vitro
and was the most effective treatment to control cobweb in crop, which suggests that it could be an efficient alternative to prochloraz-Mn (
Carrasco
et al.
, 2016b
,
2017
).

In this context, improved hygiene in growing facilities before the disease develops, as well as a better understanding of the pathogen’s behaviour, will lengthen the half-life of available fungicides by streamlining doses to prevent the occurrence of resistant outbreaks (
Schwinn & Morton, 1990
).

Alternative control methods

Due to consumer demand and environmental concerns, there is a strong pressure to reduce the use of chemical pesticides (French plan EcoPhyto 2018), which has led to the intensification of biological control in agriculture.

Finally,
Savic
et al.
(2012)
tested the antifungal activity of organic selenium against
C. dendroides.
The addition of 70-100 µg/g selenium to the substrate inhibited growth of the mycopathogen and resulted in the enrichment of basidiomes with this trace element.

Conclusion and perspectives

Certain species from the genus
Cladobotryum
may generate cobweb disease in a wide range of edible mushroom crops worldwide. Control of this pathology currently relies on prevention and hygiene measures in mushroom farms, together with chemical fungicide treatments. However, the range of available substances approved for mushroom crops is limited by the fungal nature of the host as well as by restrictive legislation. Understanding the mechanisms involved in the interaction between parasite and host is a powerful tool in the design of novel control strategies, including the production of resistant host varieties. However, many questions involving mycoparasites remain unanswered, including the pathway for infection followed by harmful species to detect and colonize the host, the molecular basis for the observed symptoms, the molecules implied in the attack on the host tissues, or the mechanisms used to overcome host defences. On the other hand, fungicide alternatives to fight cobweb disease in the form of environmentally-friendly biomolecules are being actively investigated, accompanied by a search for efficient biocontrol agents to cope with
Cladobotryum
infection. Successful biological control of the fungal diseases would satisfy the mushroom industry’s continuous efforts to minimize the use of chemicals. However, to date, no biocontrol agent has been found to be as effective as approved fungicides.

Acknowledgements

Some images have been taken by Antonio Martinez (CIES) and Jan Dijksterhuis (CBS-KNAW).